UNCONTROLLED COPY

SAM 612.04

Page 1 of 8

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 612

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix 

rhusiopathiae Vaccines

Date: February 5, 2015

Number: SAM 612.04

Supersedes: SAM 612.03, July 1, 2011

Standard Requirement: 9 CFR 113.67

Contact: Sophia G. Campbell, (515) 337-7489

Amanda L. Byersdorfer, (515) 337-7984

Approvals: /s/Larry R. Ludemann Date: 12Feb15

Larry R. Ludemann, Section Leader

Bacteriology

/s/Byron E. Rippke Date: 17Feb15

Byron E. Rippke, Acting Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 17Feb15

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
Center for Veterinary Biologics SAM 612.04

Testing Protocol Page 2 of 8

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix rhusiopathiae Vaccines

UNCONTROLLED COPY

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Record and Report of Test Results

7. References 

8. Summary of Revisions

Appendices 
Center for Veterinary Biologics SAM 612.04

Testing Protocol Page 3 of 8

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix rhusiopathiae Vaccines

UNCONTROLLED COPY

1. Introduction

This Supplemental Assay Method (SAM) establishes the titration method for the analysis of 

Erysipelothrix rhusiopathiae vaccine, live culture, to determine the colony-forming units (CFU) 

in final container samples, as prescribed in title 9, Code of Federal Regulations (9 CFR), part 

113.67. This method uses 5% bovine blood agar for determining CFUs and 1% peptone saline as 

a diluent. 

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Vortex mixer

2.1.2 Colony counter

2.1.3 Pipetting aid

2.1.4 35°± 2°C incubator

2.1.5 Laminar-flow Class II biosafety cabinet (BSC)

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 1% Peptone-saline solution (Appendix I) - National Centers for Animal 

Health (NCAH) Media #10138

2.2.2 Blood agar with 5% bovine blood (Appendix II) - NCAH Media #10006 

or as stated in the Outline of Production (OP) from the biologics manufacturer

2.2.3 E. rhusiopathiae reference culture (American Type Culture Collection

(ATCC) #19414)

2.2.4 70% ethanol

2.2.5 Sterile water in serum vials

2.2.6 Inoculum spreader
Center for Veterinary Biologics SAM 612.04

Testing Protocol Page 4 of 8

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix rhusiopathiae Vaccines

UNCONTROLLED COPY

2.2.7 Disposable syringes and needles

2.2.8 Sterile disposable pipettes

2.2.9 Sterile screw-capped culture tubes

2.2.10 Gloves and lab coat 

2.2.11 4 x 4-inch sterile gauze pads

2.2.12 Test tube rack 

2.2.13 Sharps container 

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques and preparation, proper handling, and disposal of 

biological agents, reagents, tissue culture samples, and chemicals. Personnel must also 

have knowledge of safe operating procedures and policies, as well as training in the 

operation of the necessary laboratory equipment listed in Section 2.1.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Monitor temperature of incubators according to SOPs.

3.2.3 Turn on the biosafety cabinet at least 30 minutes before starting work.

3.2.4 Label all plates with sample number or name, vial number, and dilution 

series. Label 3 plates per dilution series for each serial.

3.3 Preparation of reagents/control procedures 

3.3.1 Warm the samples and reference culture to room temperature before 

rehydrating to the appropriate volume.

3.3.2 E. rhusiopathiae reference stock culture is prepared according to the

manufacturer’s instructions.
Center for Veterinary Biologics SAM 612.04

Testing Protocol Page 5 of 8

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix rhusiopathiae Vaccines

UNCONTROLLED COPY

3.3.3 Negative and Positive Controls: Incubate 1 uninoculated plate of 5% 

bovine blood agar and 1 plate inoculated with sterile diluent along with the test 

sample plates as negative control plates. E. rhusiopathiae reference culture 

(positive control) is diluted the same as the test samples, but plated depending on 

the titer found in Section 3.3.2.

3.3.4 Store plates used for making counts at refrigerator temperature. Place 

plates to be used for counts in a 35°± 2°C incubator overnight prior to use or dry 

in a BSC before use. At the time of use, plates are no more than 3 months old.

3.4 Preparation of the sample

Samples are E. rhusiopathiae vaccines and/or combination products containing this 

fraction. Sterile purified water in serum vials in volumes specified on the product label or 

in the firm’s OP is used for rehydrating samples that are not accompanied by diluent. 

4. Performance of the Test

4.1 Remove 2 vials (or the number of vials stated in the OP for testing) of product to 

be tested and 1 vial of E. rhusiopathiae reference stock culture from the freezer or cooler 

storage and allow to warm to room temperature.

4.2 Disinfect the cap with 70% ethanol. Rehydrate the vials and allow the contents to 

reconstitute for at least 5 minutes. Shake the vials by inversion until thoroughly mixed.

4.3 Prepare a tenfold dilution series of the product by setting up a rack of 20 x 

150-mm screw-capped tubes and pipetting 9.0 mL of 1% peptone saline solution into 

each tube using a 10-mL pipette. Label the tubes 10-1to 10-xas needed.

4.4 Transfer 1.0 mL of the first sample from Section 4.2 into the first tube of 1% 

peptone saline solution by using a pipette. Cap the tube and vortex. The dilution series is 

continued by using a new pipette to transfer a 1.0 mL sample from this tube to the tube 

labeled 10-2. Repeat this method using a sterile pipette for each transfer until the required 

number of serial tenfold dilutions (refer to the firm’s OP) is attained.

4.5 Deposit 0.1 mL of the sample from the last 3 dilution points of the dilution series 

for the product onto the surface of media in Section 2.2.2 using a sterile pipette. 

4.6 Use a sterile inoculum spreader to evenly distribute the inoculum over the surface 

of the agar medium. 

4.7 Repeat Sections 4.4 through 4.6 with the second vial of product.
Center for Veterinary Biologics SAM 612.04

Testing Protocol Page 6 of 8

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix rhusiopathiae Vaccines

UNCONTROLLED COPY

4.8 Prepare 3 plates of media as in Sections 4.5 through 4.6 from each of 3 reference 

control dilutions as determined from Section 3.3.2. 

4.9 Invert all plates and incubate at 35°± 2°C for up to 72 hours. After incubation, 

count plates from each series that contains 30 to 300 CFUs. Determine the mean CFU per 

dose for the number of vials tested using the calculation listed below.

(Average Count) x (mL used to rehydrate) = CFU/dose

 (Dilution used) x (mL plated) x (Number of doses)

5. Interpretation of the Test Results

5.1 If on the initial test the CFU per dose is equal to or exceeds the required minimum 

as written in the firm’s OP, the serial or subserial is satisfactory (SAT) for bacterial count 

without additional testing.

5.2 If on the initial test the CFU per dose is less than the required minimum as written 

in the firm’s OP, the serial or subserial may be retested using double the amount of new 

vaccine samples used in the initial test, provided that if the retest is not done, the serial or 

subserial is unsatisfactory (UNSAT). Compare the firm’s OP method to this SAM when 

retesting with the new vaccine samples. If on the RT the average count of the new

vaccine samples with the firm’s OP method is less than the required minimum, the serial 

or subserial is UNSAT.

5.3 If on the retest with the new vaccine samples, the average using the firm’s OP 

method count is equal to or exceeds the required minimum, the serial is SAT.

5.4 If on the initial test the reference culture or positive control culture is not within 

the titer range determined in Section 3.3.2, but the serial being tested has a SAT result, 

the serial or subserial is a no test (NT) for bacterial count without additional testing, and 

the product is released on the results of the firm’s tests. If the reference culture is not 

within its titer range and the serial being tested is below its minimum release titer, the 

serial is retested without bias using the same number of new vaccine samples as the 

initial test. If on the initial test there is growth on the negative control plates, the serial or 

subserial is a NT for bacterial count without additional testing.

6. Record and Report of Test Results

Record and report results of the test(s) according to SOPs.
Center for Veterinary Biologics SAM 612.04

Testing Protocol Page 7 of 8

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix rhusiopathiae Vaccines

UNCONTROLLED COPY

7. References

Title 9, Code of Federal Regulations, part 113.67, U.S. Government Printing Office, 

Washington, DC.

8. Summary of Revisions

Version .04

 The Bacteriology Section Leader has been updated.

 1-6: These Sections have been clarified and updated to reflect current practices.

 Appendices: Media storage conditions have been updated.

Version .03

 The Contact information has been updated.

 References to NVSL have been changed to NCAH throughout the document.

 Appendices: Media storage conditions have been added.

Version .02

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 Amanda Byersdorfer has been added as a Contact.

 2.1: The Bunsen burner has been removed from the equipment list.

 4.8: The use of 1 plate inoculated with sterile diluent as a negative control to run 

sterility check on diluent used in testing has been added.

 4.9: The calculation to determine CFU/dose has been added.
Center for Veterinary Biologics SAM 612.04

Testing Protocol Page 8 of 8

Supplemental Assay Method for Bacterial Plate Count of Erysipelothrix rhusiopathiae Vaccines

UNCONTROLLED COPY

Appendix I

NCAH Media #10138

Peptone solution 1% + 0.5% NaCl

Bacto peptone 10.0 g

NaCl 5.0 g

QH2O 1000 mL

Autoclave for 20 minutes at 121°C. Media is stored at 2°- 5°C for up to 3 months.

Appendix II

NCAH Media #10006

Blood agar base with 5% bovine blood

Blood agar base (Difco) 40.0 g

H2O 950 mL

Autoclave for 20 minutes at 121°C.

Cool to 47°C and add: 

Defibrinated bovine blood 50 mL

Media is stored at 2°- 5°C for up to 3 months.